Substance having physiological property, method for producing the same and uses thereof

ABSTRACT

A glucoside containing isomaltol represented by formula (I):                    
     (wherein Sug represents a glucose residue or a sugar linkage composed of two or more glucose units) as an aglycon, a physiologically active substance containing the glucoside having human cytokine inducing ability increasing activity, a method for producing it, and food, medicine and feedstuff containing the substance.

TECHNICAL FIELD

The present invention relates to novel glycoside compounds, tophysiologically active substance containing them as active ingredients,to a method for producing them, and to their uses. More particularly,the present invention relates to glucosides containing isomaltol as anaglycon, to physiologically active substances containing the glucosidesas active ingredients, to a method for producing them and to their uses.

BACKGROUND ART

It is known that cultures of basidiomycete have properties of BRM(Biological Response Modifiers) such as immunopotentiation,immunological activation, or macrophage activation.

Also the present inventors have filed a patent application on an agentfor recovering productivity of cytokines such as human monocyteinterferon gamma (IFN-γ), or interleukin 12 (IL-12) using a culture ofbasidiomycete (a fungus belonging to Basidiomycetes) (Japanese PatentApplication No. Hei 11-283223).

The physiological activities contained in such a culture ofbasidiomycete have been disclosed in “Water-soluble lignin” (ExaminedJapanese Patent Publication No. Hei 6-88909), “Complex of cyctokininbased active substance composed mainly of polysaccharide andzeatin-related substance” (Examined Japanese Patent Publication No. Sho62-36009), novel polysaccharide substances (Japanese Patent ApplicationLaid-open No. Hei 8-259602) and so forth. As shown in theseapplications, basidiomycete cultures may contain several types ofphysiologically active substances. However, none of the physiologicallyactive substance has been identified by the structure in such a mannerthat the existence thereof can be unambiguously confirmed byinstrumental analysis, in particular a spectroscopic method (includingnuclear magnetic resonance or the like).

In conventional methods, plant tissue materials have been used, so thatthe cultures contain various types of organic substances. Furthermore,little information on the structure of target compounds is available.This makes it difficult to distinguish the fraction containing thetarget substance by instrumental analysis even if separations means suchas column chromatography is used. Therefore, it has been difficult toefficiently isolate and produce the component having excellentphysiological activity.

OBJECT OF THE INVENTION

It is an object of the present invention to elucidate the structure ofmain substances from among the components that bring about thephysiological activity contained in the basidiomycete cultures to whichthe present inventors have paid attention for a long time, to specifyconditions for producing the substance efficiently, and to provide usessuch as food, medicine and feedstuff utilizing the physiologicalactivity of the substance.

DISCLOSURE OF THE INVENTION

The present inventors have made extensive studies with a view to solvingthe above-mentioned problems. As a result, they have found out thatreacting a culture solution obtained by preliminarily culturing abasidiomycete with a reaction mixture obtained by reacting bran extractswith an enzyme gives rise to a substance having an increasedphysiological activity. This is in contrast to the conventional culturemethod of basidiomycete in a medium containing a plant tissue material.Also, the present inventors have identified the substance. The presentinvention has been achieved based on the discoveries.

That is, the present invention relates to novel substances having aphysiological activity, method for producing them, and their use as setforth below.

1. A glucoside containing isomaltol represented by formula

(I):

(wherein Sug represents a glucose residue or a sugar linkage composed oftwo or more glucose units) as an aglycon.

2. A glucoside according to 1 above, represented by formula (II):

(wherein n is 0 or an integer of 1 or more).

3. A glucoside according to 2 above, wherein n is 1 to 20.

4. A glucoside according to any one of 1 to 3 above, wherein theglucoside is obtained by reacting a culture solution obtained byculturing a basidiomycete with a reaction mixture of a rice branextracts solution and an enzyme, and then isolating the glucoside bycolumn chromatography.

5. A 1-O-(2-acetylfuran-3-yl)-α-D-glucopyranoside represented by formula(III):

which corresponds to the formula (II) in which n is 0.

6. A physiologically active substance comprising a glucoside accordingto 1 or 2 above as an active ingredient.

7. The substance having a physiologically activity according to 6 above,wherein the physiological activity increases human cytokine inducingability.

8. The substance having a physiological activity according to 7 above,wherein the cytokine is tumor cell necrosis factor (TNF-α).

9. The substance having a physiological activity according to 7 above,wherein the cytokine is interferon-gamma (IFN-γ).

10. The substance having a physiological activity according to 7 above,wherein the cytokine is interleukin-2 (IL-2).

11. A method for producing a glucoside represented by formula (I):

(wherein symbols have the same meanings as defined in 1 above) having aphysiological activity, comprising the step of reacting a culturesolution obtained by culturing a basidiomycete with a reaction mixtureobtained by reaction between a rice bran extracts solution and anenzyme, and isolating the glucoside.

12. A method for producing a glucoside represented by formula (I) in 1above, having a physiological activity, comprising the steps of reactinga culture solution obtained by culturing a basidiomycete with a reactionmixture obtained between a rice bran extracts solution and an enzyme,and isolating the glucoside by column chromatography using as an index acomponent showing (1) a peak attributable to methyl hydrogen of methylketone by ¹H-NMR spectrum and a peak attributable to hydrogen atoms atthe 4,5-positions of a 2,3-di-substituted furan ring characteristic ofAB system, and (2) 6 peaks attributable to glucopyranose and 6 peaksattributable to isomaltol by ¹³C-NMR.

13. A food containing a substance having a physiological activityaccording to any one of 6 to 10 above.

14. A medicine containing a substance having a physiological activityaccording to any one of 6 to 10 above.

15. A feedstuff containing a substance having a physiological activityaccording to any one of 6 to 10 above.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a graph showing the results of measurement of TNF-αconcentration in the supernatant of spleen cell culture afteradministration of the substance A in the present invention (compound offormula (III)), substance B in the present invention (compound offormula (I)), and crude product according to the present invention (CR).

FIG. 2 is a graph showing the results of measurement of IFN-αconcentration in the supernatant of spleen cell culture afteradministration of the inventive substance A in the present invention(compound of formula (III)), inventive substance B in the presentinvention (compound of formula (I)), and crude product according to thepresent invention (CR).

FIG. 3 is a graph showing the results of measurement of IL-2concentration in the supernatant of spleen cell culture afteradministration of the inventive substance A in the present invention(compound of formula (III)), inventive substance B in the presentinvention (compound of formula (I)), and crude product according to thepresent invention (CR).

FIG. 4 is a graph showing the results of measurement of TNF-αconcentration in the supernatant of macrophage culture afteradministration of substance A in the present invention (compound offormula (III)), substance B in the present invention (compound offormula (I)) and crude product according to the present invention (CR).

FIG. 5 is a graph showing the results of measurement of IFN-γconcentration in the supernatant of macrophage culture afteradministration of substance A in the present invention (compound offormula (III)), substance B in the present invention(compound of formula(I)), and crude product according to the present invention (CR).

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be illustrated in detail.

(A) Glucoside Compounds

The glucoside compounds of the present invention are novel glucosideshaving isomaltol as an aglycon, represented by formula (I)

obtained by reacting a culture solution obtained by culturing abasidiomycete with a reaction mixture obtained by a bran extract and anenzyme (hereinafter sometimes abbreviated as “rice bran extractssolution-enzyme reaction mixture”) and isolating by columnchromatography (details will be described hereinbelow).

The isomaltol is a substance represented by formula:

which is used as a component of a perfume.

As for the glycoside having isomaltol as an aglycon, there is an examplethat refers to galactoside (glycoside of which the sugar moiety isgalactose (J. Bartulin et al., J. Heterocycle Chem., 29, 1017 (1992),“Syntheses of 2-Acetyl-3-hydroxy-1-n-propylpyrrole from Isomaltol and1-n-Alkyl-3-hydroxy-2-methyl-4-pyridones from Maltol”). However, noreport has been made on glucoside (which is a glycoside with the sugarmoiety composed of glucose units), and functions of such glucoside areunknown.

In the case where Sug contains 2 or more glucose units, the length ofsugar linkage is not particularly limited. According to the method ofthe present invention, mixtures of glucosides with n varying from 1 to20 occupy about 10% of the solid reaction product. The sugar linkage(Sug) may contain α(1→6) bond or other bonds as a part thereof. Also itmay contain branched chain. However, it is a linear sugar linkage mainlycomprising α(1→4) bonds.

A typical structure of novel glycoside compound is represented byformula (II):

(wherein n is 0 or an integer of 1 or more).

For example, novel 1-O-(2-acetylfuran-3-yl)-α-D-glucopyranosiderepresented by formula (III):

having one glucose unit, which corresponds to the formula (II) above inwhich n is 0, can be isolated by the method of the present invention orprepared by chemical synthesis.

(B) Physiologically Active Substance

The novel glucoside compound of the present invention mentioned abovehas excellent physiological activities.

The physiological activities include immunopotentiation/activation,macrophage activation and the like properties, in particular improvementof cytokine inducing ability of human cells.

Examples of the cytokine induced by the physiological active substanceof the present invention include tumor necrosis factor (TNF-α),interferon gamma (IFN-γ), interleukin-2 (IL-2) and so forth. However, itis not limited to these.

The tumor necrosis factor TNF-α is a protein that has activity ofkilling tumor cells and is a cytokine produced systemically or locallyin response to infection, wound or immunological induction. Interferon(IFN) is an antiviral protein produced upon receiving stimulation byvirus or nucleic acid and secreted outside cells. It is known thatinterferons exhibit not only antiviral activity but also variousactivities such as antitumor activity and activation of immune systems.IFN-γ is produced by T cells and so forth. Interleukin-2 (IL-2) haseffects of degeneration or eradication of tumors.

(C) Production and Isolation Methods

The compounds represented by the formula (I) above having thephysiological activity as described above are novel substances that havenot been reported yet. The present inventors have confirmed that thecompounds are not present in cultures of basidiomycete or in the ricebran extracts solution-enzyme reaction mixture, and that they are notproduced when rice bran is added to the medium from the beginning ofculture. They also confirmed that the compounds could be produced onlyin a solution obtained by reacting the culture of a basidiomycete withthe rice bran extracts solution-enzyme reaction mixture.

Conventionally, cultivation of a basidiomycete has been performed inmedia containing plant tissue materials. However, it is beyondexpectation that reaction between a culture solution obtained bypreliminarily culturing basidiomycete and a rice bran extractssolution-enzyme reaction mixture can give rise to substances having highphysiological activities.

(C-1) Basidiomycete Culture Solution

The basidiomycete culture solution used in the process of producing thephysiologically active substances of the present invention is a culturesolution obtained by culturing a basidiomycete in a liquid mediumsuitable for its growth.

Examples of carbon source in the liquid medium include glucose, sucrose,maltose, saccharose, high grade white sugar, black sugar, molasses,waste molasses, malt extracts and so forth.

Examples of nitrogen source includes, meat extracts, peptone, glutenmeal, soybean powder, dry yeast, yeast extracts, ammonium sulfate,ammonium tartrate, urea and so forth. In addition, inorganic salts suchas sodium salts, magnesium salts, manganese salts, iron salts, calciumsalts and phosphoric acid salts, vitamins such as inositol, vitamin B1hydrochloride, L-asparagine, and biotin may optionally be added.

The composition of preferred liquid medium may vary depending on thestrain of basidiomycete but may be comprised, for example, by maltextracts (1 to 5%), yeast extracts (0.1 to 0.5%), and ammonium tartrate(0.1 to 0.4%).

Examples of the basidiomycete used in the present invention includeLentinum edodes, Agaricus bisporus, Grifola frondosa, Phoriota nameko,Pleurotus ostreatus, Flammulina velutipes, Ganoderma lucidum,Auricularia auricura, Ganoderma applanalum, Coriolus lucidum, Grifolaumbellate, Shizophyllum commune, Volvariella volvaceae and so forth.Particularly preferred are Lentinus edodes and Grifola frondosa.

The culture is performed according to aeration culture for ordinarymedium temperature microbes, i.e., at pH 2 to 6 and at a temperature of10 to 45° C., preferably 15 to 30° C. The culture time may varydepending on the type and amount of the microbe and usually it lasts for4 to 20 days, preferably 6 to 12 days.

After the cultivation, solid-liquid separation is performed and theliquid components are used for subsequent reaction with the rice branextracts solution-enzyme reaction mixture.

(C-2) Rice Bran Extracts Solution-enzyme Reaction Mixture

The rice bran extracts solution is an aqueous solution obtained bystirring a certain amount of rice bran in an equivalent amount or more(for example, 2 to 10 folds) of hot water. On this occasion, it ispreferred to use pressurized water at about 105 to about 130° C.Subsequently, rice bran is reacted by addition of an enzyme agent andsubjected to solid-liquid separation using a decanter or the like toobtain a rice bran extracts solution-enzyme reaction mixture.

As the enzyme agent, a carbohydrate hydrolase such as α-amylase,cellulase or pectinase may be used alone or in combination of two ormore of them. A proteolytic enzyme such as protease may be used incombination. The enzyme reaction is performed under conditions where theenzyme activity is not damaged, that is, at about 40° C. or higher andabout 90° C. or lower, preferably about 40 to about 70° C., for about 1to about 24 hours, preferably about 3 to about 12 hours.

(C-3) Reaction between Basidiomycete Culture Medium and Rice BranExtracts Solution-enzyme Reaction Mixture

The reaction between a basidiomycete culture and a rice bran extractssolution-enzyme reaction mixture is carried out preferably at 40° C. orhigher and 90° C. or lower and more preferably 40 to 70° C. Usually, theboth reactants are mixed and allowed to react for 8 to 24 hours. Then,the whole liquid is heated at 120° C. or higher under pressure toinactivate the enzymes and the like in the liquid and optionallyseparated and purified.

The separation and purification methods are not particularly limited andvarious separation methods may be applied.

In the case of conventional basidiomycete-derived physiologically activesubstances, selections of constitution of column and an eluent have beendetermined in a trial-and-error manner by way of color reactions orbiological examinations. The color reactions allow ambiguousinterpretation or biological examinations that takes a long time forjudgment since few data on the chemical structure of the objectivecompound have been available. In contrast, in the present invention, thechemical structure of the objective physiologically active substance isalready clarified and selection of the constitution of column and eluentcan be easily made according to the hydrophilicity, molecular weight andother properties of the substance based on the structural formulathereof. Further, objective fractions can be quickly distinguished byusing the following spectroscopic characteristics as indices.

(1) The ¹H-NMR spectrum shows a peak attributable to the methyl-hydrogenof methyl ketone and AB system peaks attributable to the hydrogen atomsat the 4,5-positions of a 2,3-di-substituted furan ring.

(2) The ¹³C-NMR spectrum shows 6 peaks attributable to glucopyranose and6 peaks attributable to isomaltose.

The peak attributable to the methyl-hydrogen of methyl ketone is foundnear δ2.48 (3H, s). The peaks attributable to the hydrogen atoms at the4,5-positions of 2,3-di-substituted furan ring are recognized as ABsystem near δ6.78 and δ7.67 (both values were measured in deuteratedmethanol).

Besides, reference may be made to infrared absorptions at 3000 to 3700cm⁻¹ (hydroxyl group), 1660 cm⁻¹ (conjugated carbonyl group) and 1584cm⁻¹ (double bond).

(E) Uses

The physiologically active substance of the present invention uses safematerials that have conventionally been provided as esculent materials(rice bran extracts solution and edible basidiomycete culture medium)and there are little possibilities of being contaminated with toxic sideproducts. Therefore, the physiologically active substance of the presentinvention itself can be widely utilized as health food, medicine,reinforcing food, feedstuff and so forth.

When it is used for these uses, for example, as a health food, thephysiologically active substance obtained in the above process (C-3) isconcentrated or purified depending on the purpose. The physiologicallyactive substance of the present invention is relatively stable to heat.However, it is preferred that lyophilization be used for theconcentration and drying. The physiologically active substance of thepresent invention may be formulated using a food additive, an excipientor the like.

The medicinal uses may include medicines for humans and medicines foranimals. When the inventive products are administered as a medicine, thephysiologically active substances of the formula (I) above may be usedas they are or as a medical composition obtained by adding it to anon-toxic, inert carrier that is pharmaceutically acceptable. As thecarrier, solid, semi-solid or liquid diluents, filler and auxiliariesfor other formulations and so forth are used.

The medicines may be administered by intravenous administration, oraladministration, tissue administration, topical administration(percutaneous administration, etc.) or perrectal administration. Theformulation may be of any form that is suitable for the aboveadministration methods.

Uses as the feedstuff includes feedstuff for livestock and poultry suchas cows, pigs, hens and sheep, feedstuff for fish culture for fish orcrustaceans, and feedstuff for pets such as dogs, cats and others.

In recent years, the effects of minimizing the problems caused by use oflarge amounts of antibiotics, which attention has been paid to in thefield of animal husbandry, can be expected. The above physiologicallyactive substances may be orally administered alone but it is preferableto use them together with various glycosides, proteins, lipids, fibers,vitamins, and minerals and so forth as feedstuff in the form of dusts orpellets. The types of lipids and fibers are not particularly limited butany substance that can be used as feedstuff, such as fish, meat bonepowder, soybean meal, alfalfa meal, and bran.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the physiologically active substance of the presentinvention will be explained in detail by production examples andphysiological activity assay results.

The physiological activity assays were performed by forciblyadministering 1 g/kg of a sample to 6-week age male C57/BL6 mice for 1week by oral route. Spleen cells and peritoneal exudate cells (PEC) werecollected after sacrificing the animals. They were cultivated andassayed TNF-α, IFN-γ, and IL-2.

a) Cultivation of Spleen Cells

The collected mouse spleen was homogenized in DMEM medium and thehomogenate was centrifuged at 4° C. (1,000 rpm, 5 minutes). To this wasadded ASK lysis buffer and the mixture was incubated (37° C., 3 minutes)in order to remove erythrocytes, followed by centrifugation (1,000 rpm,5 minutes). To the spleen cells thus collected was added ConA at a finalconcentration of 10 μg/ml and cultivated in a microplate. After 15hours, the culture supernatant was collected for assaying TNF-α, IFN-γ,and IL-2.

b) PEC Cultivation

PEC (peritoneal exudate cells) collected from mouse abdominal cavitywith physiological saline were centrifuged (1,000 rpm, 10 minutes) tocollect cells, which were used as macrophages. To the macrophages LPS(lipopolysaccharide) was added at a final concentration of 100 ng/ml andthe cells were cultivated. After 4 hours, TNF-α, IFN-γ, and IL-2 in theculture supernatant were assayed.

For both a) and b) above, a kit for assaying cytokine (Wako PureChemical Industries, Ltd.) was used.

EXAMPLE 1

Confirmation of Physiological Active Substance

In a liquid medium having the composition containing malt extracts (2%),yeast extracts (0.25%) and ammonium tartrate (0.2%), Shiitake mushroom(Lentinus edodes) was cultivated with aeration at 24° C. for 10 days toprepare a basidiomycete culture solution.

On the other hand, rice bran was dispersed in 5-fold (mass) water andstirred at a liquid temperature of 120° C. for 20 minutes to effectextraction. Thereafter, an enzyme agent composed of α-amylase andpectinase was added to thereto, and reaction followed at 60° C. for 120minutes. Then, the reaction mixture was subjected to solid-liquidseparation and the liquid portion was recovered to prepare a rice branextracts solution-enzyme reaction mixture.

Neither the basidiomycete culture solution nor rice bran extractssolution-enzyme reaction mixture exhibited any physiological activity.

On the other hand, the basidiomycete culture solution and the rice branextracts solution-enzyme reaction mixture obtained by the aboveprocedures were mixed together and the resultant mixture was stirred at60° C. for 20 hours for reaction. Then, the whole liquid was heated at120° C. or higher for 20 minutes to inactivate the enzymes and so forthin the liquid, the reaction mixture was concentrated, and 80 ml of thereaction mixture (moisture content:: 75%) was eluted with H₂O, then withmethanol through a 500-ml DIAION HP-20 column. As a result,physiological activity was observed in the eluate. Therefore, thisprocedure was repeated twice to obtain a brown fraction (5.6 g).

Further, the fraction was subjected to detection of physiologicallyactive components by various column chromatographic analyses. That is,the fraction was subjected to silica gel chromatography (Kiesel,produced by Merck & Co., Inc., 125-mesh, 50 g) and subsequently to ODSbased reversed phase gel column chromatography (about 100 ml in volume)to obtain white powdery physiologically active component (40 mg).

The obtained substance had the following physical properties.

(i) Mass Spectrometry (Secondary Ion Mass Spectrometry (SIMS))

Negative ionization measurement showed a molecular ion peak at m/z: 287and positive ionization measurement showed a molecular ion peak of m/z:289. As a result, the molecular weight of the present compound was foundto be 288. Further, the above results together with the results ofhigh-resolution mass spectrometry of these ion peaks suggested that themolecular formula of the compound would be C₁₂H₁₆O₈ (degree ofunsaturation: 5).

(ii) IR Spectrum

Absorptions were observed at 3000 to 3700 cm⁻¹ attributable to ahydroxyl group, at 1660 cm⁻¹ attributable to a conjugated carbonyl groupand at 1584 cm⁻¹ attributable to a double bond.

(iii) ¹H-NMR Spectrum (in Deuterated Methanol, 270 MHz)

There were observed signals at δ2.48 (3H, s) attributable to methylketone and at δ3.4-5.6 attributable to α-D-glucopyranose ring, as wellas peaks at δ6.78 and δ7.67 attributable to an AB system having acoupling constant of 1.9 Hz.

(iv) ¹³C-NMR Spectrum (in Deuterated Methanol, 67.5 MHz, DEPT MeasuringMethod)

Twelve (12) signals were observed. That is, besides a group of sixsignals attributable to α-D-glucopyranose ring, six signals attributableto aglycon moieties were observed at δ27.4 (primary), δ105.9 and 139.3(each being tertiary), δ148.5, 154.3, and 187.4 (each being quaternary).

The low magnetic field AB system in ¹H-NMR is considered to becharacteristic to the hydrogen signal at the 4,5-positions of2,3-di-substituted furan ring in view of its coupling constant being 1.9Hz. The degree of unsaturation of the molecular formula being 5corresponds to one pyranose ring, two double bonds, one carbonyl groupand one furan ring. From the above information, the obtained substancewas confirmed to be a novel substance(1-O-(2-acetylfuran-3-yl)-α-D-glucopyranoside, hereinafter referred toas “substance A”) represented by the formula below.

Confirmation of Production Process of Substance A

In the same manner as described above, the rise and fall of thesubstance A that would exist in the basidiomycete culture solution, ricebran extracts solution-enzyme reaction mixture, the reaction mixturebetween the above two and so forth were followed up. As a result, it wasconfirmed that the substance A was produced only in the reaction mixturebetween the basidiomycete culture solution and the rice bran extractssolution-enzyme reaction mixture. No substance A existed in thebasidiomycete culture solution or in the rice bran extractssolution-enzyme reaction mixture. It revealed that the substance A isproduced only by reaction between the two.

EXAMPLE 2

Chemical Synthesis of Substance A

In accordance with the example of reaction in which galactosylisomaltolis chemically derived from lactose as a starting material as disclosedin J. Bartulin et al., J. Heterocycle Chem., 29, 1017 (1992), “Synthesesof 2-Acetyl-3-hydroxy-1-n-propylpyrrole from Isomaltol and1-n-Alkyl-3-hydroxy-2-methyl-4-pyridones form Maltol,” substance A waschemically derived using maltose in place of lactose.

(a) Chemical Derivation Reaction

In a 50-ml eggplant type flask, 3.60 g (0.01 mol) of maltose monohydratewas weighed and 3.0 ml of anhydrous ethanol was added thereto. Themixture was stirred vigorously with a stirrer contained therein todisperse maltose. On this occasion, the state in which an undissolvedportion of the maltose monohydrate was stirred and uniformly dispersedthroughout the anhydrous ethanol without localization was maintained.While vigorously stirring the mixture, 1.0 ml of piperidine was addedthereto and uniformly mixed therewith. Then, 0.44 ml of acetic acid wasslowly dripped over about 15 minutes using a measuring pipette. In asilicone oil bath adjusted to 78° C., the eggplant type flask equippedwith a water stream condenser was placed and reaction is allowed toproceed. When the heat distributed throughout the flask throughout andboiling started in portions thereof, about 0.5 ml of triethylamine wasadded. After the placement of the flask, maltose monohydrate began to bedissolved due to the heat and accordingly the solution turned fromyellow to brown. After about 12 hours, the same volume as above oftriethylamine was added to keep the liquid basic. After 24 hours, theflask was taken out from the refluxing apparatus and the temperature ofthe flask was returned to room temperature before purification operationcould be proceeded. Then the reaction product was intense brown tar-likesubstance in nature.

The reaction product was subjected to TLC analysis using a plate (Merck,Kieselgel60F254) with a solvent (chloroform/methanol/water (65:25:4)mixed solution). As a result, a spot having an absorption whenirradiated by ultraviolet rays with a main wavelength of 254 nm wasobserved at the same Rf value (about 0.5) as substance A. When it issprayed with an anisaldehyde-sulfuric acid coloring reagent and heated,the spot turned yellow and further intense blue.

(b) Purification

(b-1) Crude purification with LH-20 gel

A necessary volume of LH-20 gel was preliminarily passed throughmethanol/water (1:2) to equilibrate it and the reaction mixture mixedwith a minimum amount of the same solvent was charged thereto. Thereaction mixture was developed and fractionated with the same solventand the fractions containing the objective compound were combined. Thefraction obtained was evaporated and concentrated to dryness to obtain abrown oily substance (0.49 g).

(b-2) Purification by Silica Gel Column Chromatography

Sample was charged and developed in a column prepared using silica gelof about 200 mesh in an amount of 20 to 30 times the weight of thesample. Chromatography was performed again with water-saturated ethylacetate/methanol (3:1) and then rechlomatographed with achloroform/methanol/water mixed solution (a saturation amount of waterwas added to 4:1 chloroform/methanol). The purified fraction wasevaporated and concentrated to dryness to obtain a yellow resinoussubstance (0.49 g)

(b-3) Crystallization

The yellow resinous substance was crystallized from methanol to obtaincolorless needle crystals (0.37 g).

The substance thus obtained showed the same behavior on chromatographyand NMR spectrum as those of the substance A.

EXAMPLE 3

Existence of the substance A in the reaction mixture between thebasidiomycete culture solution and the rice bran extractssolution-enzyme reaction mixture suggested the possibility that thephysiologically active substance in the basidiomycete culture solutioncould comprise glucosides having isomaltol structures at the reducingterminals thereof with sugar linkages of different lengths.

Accordingly, the basidiomycete culture solution and the rice branextracts solution-enzyme reaction mixture were prepared by the sameprocedures as in Example 1 and reacted at 60° C. for 20 hours bystirring. Then the entire reaction mixture was heated to 120° C. orhigher for 20 minutes to inactivate the enzymes and so forth in thereaction mixture. Thereafter, effective components having highermolecular weights were searched using the following spectroscopiccharacteristics as indices.

(a) ¹H-NMR spectrum shows a peak attributable to methyl hydrogen ofmethyl ketone and an AB system peak attributable to hydrogen atoms atthe 4,5-positions of a 2,3-di-substituted furan ring, and

(b) ¹³C-NMR spectrum shows 6 peaks attributable to glucopyranose and 6peaks attributable to isomaltol.

More particularly, in a column for chromatography (DIAION HP-20, volume:about 500 ml) was charged 80 ml (20 g as solids) of the aboveconcentrate and then eluted with 1.5 liters of water. The eluate wasdried under vacuum to obtain 18 g of powder. This fraction was dissolvedin 45 ml of water and the obtained solution was dispensed in twocentrifuging tubes each in an amount of 22.5 ml. While stirring eachsolution, a 4-fold volume, i.e., 90 ml of ethanol was slowly dripped toeach solution, which then was centrifuged at 3,000 rpm for 10 minutes.The obtained supernatants were combined, concentrated and freeze-driedto obtain powder (12 g). The powder was dissolved in 50 ml of water andthe solution was purified through column chromatography (DOWEX 50W-X8(H⁺ type)). 10.8 g of the obtained water-eluted portion was furtherpurified through column chromatography (AMBERLITE IRA-400 (CO₃ ²⁻type)), concentrated and freeze-dried to obtain 8.64 g of water-elutedpowder. This was further purified through column chromatography(SEPHADEX G-15) to obtain 2.16 g of a component that satisfies therequirements (a) and (b) above.

The obtained substance had the following physical properties.

(i) ¹H-NMR Spectrum (in Deuterated Water, 270 MHz)

Weak, broad proton signals presumed to be attributable to AB system on afuran ring were detected at δ6.85 and δ7.35.

(iv) ¹³C-NMR Spectrum (in Deuterated Water)

Weak signals presumed to be attributable to isomaltol were observed atδ26 (—C(O)CH₃), 111 (C-4), 141 (C-3), 145 (C-5), and 188 (—C(O)—).

From the above, the obtained substance is presumed to have a structurerepresented by the following formula:

This substance (hereinafter referred to as “substance B”) is a mixtureof compounds between isomaltol and oligosaccharides (composed ofD-glucose molecules with different degrees of polymerization) (in theabove general formula (II), n=2 to 20). It has an average molecularweight of 850 as obtained based on a calibration curve prepared inadvance. The oligosaccharides include mainly glucose molecules withα-1,4 glycoside bonds and partially those with α-1,6 bonds or otherbonds.

In the same manner as in Example 1 (2), the rise and fall of thesubstance B that would exist in the basidiomycete culture solution, ricebran extracts solution-enzyme reaction mixture, the reaction mixturebetween the above two and so forth were followed up. As a result, it wasconfirmed that the substance B was produced only in the reaction mixturebetween the basidiomycete culture solution and the rice bran extractssolution-enzyme reaction mixture. No substance B existed in thebasidiomycete culture solution or in the rice bran extractssolution-enzyme reaction mixture. It revealed that the substance B isnot produced before the both of them could be reacted. As will be shownin Example 4, it was confirmed that the substance B has a physiologicalactivity.

EXAMPLE 4

The physiological activities of the substance A obtained in Example 1and the substance B obtained in Example 3 were compared.

FIGS. 1 to 3 illustrate the results of measurement of concentrations ofcytokines (TNF-α, IFN-γ and IL-2) in the supernatant of a spleen cellculture after administration of the substance A, substance B and crudeconcentrate. FIGS. 5 and 6 illustrate concentrations of cytokines (TNF-αand IFN-γ) in the supernatant of a macrophage culture. As a result, itrevealed that the substance A showed considerable cytokine inductionability and the substance B showed superior effects in IFN-γ inductionability to the crude concentrate.

Concerning macrophage, it was observed that the substance A had highcytokine induction ability.

INDUSTRIAL APPLICABILITY

According to the present invention, it has been found that the componenthaving a strong physiological activity contained in an amount of about10% in the powder obtained by concentrating to dryness the reactionmixture between the basidiomycete culture solution and the rice branextracts solution-enzyme reaction mixture includes a series of compoundshaving a glucoside structure containing isomaltol as an aglycon. Thecompounds can be purified so as to have a high purity by a conventionalmeans such as column chromatography using spectroscopic characteristicsas indices.

Therefore, according to the present invention, it is possible to obtaina physiologically active substance with less contamination as comparedwith the conventional process for producing a physiologically activesubstance derived from a basidiomycete culture that uses a plant tissuematerial. The product is useful as food, feedstuff and medicine.

The glycosides that are the physiologically active substances of thepresent invention are not contained in the basidiomycete culturesolutions. They are not produced merely by reacting rice bran extractswith an enzyme. The reaction between the basidiomycete culture solutionand the rice bran extracts solution-enzyme reaction mixture isindispensable.

From the above, the substance A can be utilized as an index componentfor a process for producing medicines and for production managementtherefor. That is, when the substance A is detected, it may be presumedthat the reaction between the basidiomycete and the rice bran extractssolution-enzyme reaction mixture is used in producing such products. Theproduction amount of the substance A may vary depending on the methodand conditions for cultivating a basidiomycete, conditions for preparingrice bran extracts solution-enzyme reaction mixture (kind of enzymeused, conditions of enzyme reaction and so forth), conditions for thereaction between the two. However, production management for novelsubstances having a physiological activity of the present invention canbe realized using the substance A as an index.

What is claimed is:
 1. A glucoside containing isomaltol represented byformula (I):

wherein Sug represents a sugar linkage composed of two to twenty glucoseunits as an aglycon.
 2. A glucoside according to claim 1, represented byformula (II):

wherein n is an integer of 1 to
 20. 3. A glucoside according to claim 1or 2, wherein said glucoside is obtained by reacting a culture solutionobtained by culturing a basidiomycete with a reaction mixture of a ricebran extracts solution and an enzyme, and then isolating said glucosideby column chromatography.
 4. A1-O-(2-acetylfuran-3-yl)-α-D-glucopyranoside represented by formula(III):

which corresponds to the formula (II) in which n is
 0. 5. Aphysiologically active substance comprising a glucoside according toclaim 1 or 2 as an active ingredient.
 6. The substance having aphysiologically activity according to claim 5, wherein saidphysiological activity is an activity of increasing human cytokineinducing ability.
 7. The substance having a physiological activityaccording to claim 6, wherein said cytokine is tumor cell necrosisfactor (TNF-α).
 8. The substance having a physiological activityaccording to claim 6, wherein said cytokine is interferon-gamma (IFN-γ).9. The substance having a physiological activity according to claim 6,wherein said cytokine is interleukin-2 (IL-2).
 10. A food containing asubstance having a physiological activity according to claim
 5. 11. Amedicine containing a substance having a physiological activityaccording to claim
 5. 12. A feedstuff containing a substance having aphysiological activity according to claim 5.